Antigenic and genetic characterization of the novel influenza A H1N1 virus indicated that the virus contained a unique combination of gene segments from North American and Eurasian lineages of swine influenza viruses. The viruses isolated from human cases of the 2009 pandemic H1N1 (pH1N1) virus infection were antigenically homogeneous and antigenically similar to North American swine H1N1 viruses, but distinct from seasonal human influenza A H1N1 viruses. The swine-origin hemagglutinin (HA) was closely related to the HAs of pre-1947 human H1N1 viruses but highly divergent from the HAs of recently circulating H1N1 strains. Consequently, prior exposure to pH1N1-like viruses was mostly limited to individuals over the age of about 60 years. In collaboration with colleagues at the University of Rochester, we related age and associated differences in immune history to the B cell response to an inactivated monovalent pH1N1 vaccine given intramuscularly to subjects in three age cohorts: 18 to 32 years, 60 to 69 years, and>70 years. In FY13, we completed the analysis of the samples collected from the clinical trial. The day 0 pH1N1 specific hemagglutination inhibition (HAI) and microneutralization (MN) titers were generally higher in the older cohorts, consistent with greater prevaccination exposure to pH1N1-like viruses. Most subjects in each cohort responded well to vaccination, with early formation of circulating virus specific antibody (Ab)-secreting cells and >4-fold increases in HAI and MN titers. However, the response was strongest in the 18- to 32-year cohort. Circulating levels of HA stalk-reactive Abs were increased after vaccination, especially in the 18- to 32-year cohort, raising the possibility of elevated levels of cross reactive neutralizing Abs. In the young cohort, an increase in MN activity against the seasonal influenza virus A/Brisbane/59/07 after vaccination was generally associated with an increase in the anti- Brisbane/59/07 HAI titer, suggesting an effect mediated primarily by HA head-reactive rather than stalk-reactive Abs. Our findings support recent proposals that immunization with a relatively novel HA favors the induction of Abs against conserved epitopes. They also emphasize the need to clarify how the level of circulating stalk-reactive Abs relates to resistance to influenza. In collaboration with colleagues at Stanford University and the University of Rochester, we also compared the heterovariant B-cell response induced by the monovalent inactivated 2009 pandemic influenza A virus subtype H1N1 vaccine with that induced by the 2009 seasonal trivalent influenza vaccine (sTIV) containing a seasonal influenza A virus subtype H1N1 component in young and elderly adults. Plasmablast-derived polyclonal antibodies (PPAb) from young and elderly recipients of pH1N1 vaccine or sTIV were tested for binding activity to various influenza antigens. In pH1N1) vaccine recipients, the PPAb titers against homotypic pH1N1 vaccine were similar to those against the heterovariant seasonal H1N1 vaccine and were similar between young and elderly subjects. The PPAb avidity was higher among elderly individuals, compared with young individuals. In contrast, the young sTIV recipients had 10-fold lower heterovariant PPAb titers against the pH1N1 vaccine than against the homotypic seasonal H1N1 vaccine. In binding assays with recombinant head and stalk domains of hemagglutinin, PPAb from the pH1N1 recipients but not PPAb from the sTIV recipients bound to the conserved stalk domain. Thus, the pH1N1 vaccine induced production of PPAb with heterovariant reactivity, including antibodies targeting the conserved hemagglutinin stalk domain. In addition to the trivalent inactivated vaccine (TIV), administered by intramuscular injection, a live attenuated influenza virus (LAIV) vaccine is licensed for healthy adults 2-49 years of age. This vaccine is administered by nasal spray. Neutralizing antibody in the serum has been found to be a correlate of protection for TIV, but the immune correlates of protection for LAIV are not known. Defining the origin and nature of transcriptional responses to LAIV in upper respiratory tract will be a highly informative first step in a systems approach toward understanding the molecular basis of viral replication restriction and the regulation of the local mucosal immune responses following LAIV administration. In FY13, in collaboration with colleagues at Stanford University, we undertook a natural history study using a systems biology approach to identify LAIV replication niches among a variety of URT cell types and characterize the host immune response to LAIV. Data analysis is in progress.